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Difco bacto agar
DHHC20 expression induces transformation of NIH/3t3 cells. (A) Suspensions of each clone in exponential growth phase were plated in <t>DMEM</t> containing 0.33% <t>Bacto-Agar</t> overlaid in 35 mm plates with 0.6% agar gel. The cells were incubated for 21 days and
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1) Product Images from "DHHC20: a human palmitoyl acyltransferase that causes cellular transformation"

Article Title: DHHC20: a human palmitoyl acyltransferase that causes cellular transformation

Journal: Molecular membrane biology

doi: 10.3109/09687681003616854

DHHC20 expression induces transformation of NIH/3t3 cells. (A) Suspensions of each clone in exponential growth phase were plated in DMEM containing 0.33% Bacto-Agar overlaid in 35 mm plates with 0.6% agar gel. The cells were incubated for 21 days and
Figure Legend Snippet: DHHC20 expression induces transformation of NIH/3t3 cells. (A) Suspensions of each clone in exponential growth phase were plated in DMEM containing 0.33% Bacto-Agar overlaid in 35 mm plates with 0.6% agar gel. The cells were incubated for 21 days and

Techniques Used: Expressing, Transformation Assay, Incubation

2) Product Images from "Blocking the PI3K/AKT pathway enhances mammalian reovirus replication by repressing IFN-stimulated genes"

Article Title: Blocking the PI3K/AKT pathway enhances mammalian reovirus replication by repressing IFN-stimulated genes

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2015.00886

Modulation of reovirus replication by the PI3K/Akt pathway. Serum-starved A549 cells were pre-incubated with increasing concentrations of LY294002, wortmannin or DMSO for 1 h, and they were then infected with MPC/04 and B/03 at a MOI of 5 in the presence of each drug for 24 h. The total RNA of the cells was prepared, and S4 gene expression was analyzed by qPCR (A) . Cells and cell supernatants were harvested together, and subjected to a single freeze/thaw step for analysis of viral titers (B) . Expression of Akt1 was determined by qPCR analysis and Western blot analysis using Akt1- and GAPDH-specific antibodies (C) . Effect of inhibition of Akt1 expression on virus replication was also evaluated by plaque sizes in A549 cells (D) . A549 cells were transfected with 4 μg/well of shAkt or shCtrl for 24 h, and the cells were then infected with MPC/04 and B/03. After 1 h incubation, the monolayers were covered with 2 ml of 1% Bacto Agar and 2 × MEM containing 10 μg/ml TLCK-treated α-CHT. Plaques were photographed 4 days later. (E) A549 cells were transfected with 2 μg/well of shAkt or shCtrl for 12 h, and the cells were then infected with MPC/04 or B/03 at a MOI of 5. Cell supernatants were harvested at the indicated time point and titrated on L929 cells for the plaque assay. The data shown in (A–C) and (E) represent the results of three independent experiments. * p
Figure Legend Snippet: Modulation of reovirus replication by the PI3K/Akt pathway. Serum-starved A549 cells were pre-incubated with increasing concentrations of LY294002, wortmannin or DMSO for 1 h, and they were then infected with MPC/04 and B/03 at a MOI of 5 in the presence of each drug for 24 h. The total RNA of the cells was prepared, and S4 gene expression was analyzed by qPCR (A) . Cells and cell supernatants were harvested together, and subjected to a single freeze/thaw step for analysis of viral titers (B) . Expression of Akt1 was determined by qPCR analysis and Western blot analysis using Akt1- and GAPDH-specific antibodies (C) . Effect of inhibition of Akt1 expression on virus replication was also evaluated by plaque sizes in A549 cells (D) . A549 cells were transfected with 4 μg/well of shAkt or shCtrl for 24 h, and the cells were then infected with MPC/04 and B/03. After 1 h incubation, the monolayers were covered with 2 ml of 1% Bacto Agar and 2 × MEM containing 10 μg/ml TLCK-treated α-CHT. Plaques were photographed 4 days later. (E) A549 cells were transfected with 2 μg/well of shAkt or shCtrl for 12 h, and the cells were then infected with MPC/04 or B/03 at a MOI of 5. Cell supernatants were harvested at the indicated time point and titrated on L929 cells for the plaque assay. The data shown in (A–C) and (E) represent the results of three independent experiments. * p

Techniques Used: Incubation, Infection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Inhibition, Transfection, Plaque Assay

Role of IFN-stimulated genes in PI3K/AKT-mediated modulation of virus replication. (A) A549 cells were infected with MPC/04 and B/03 at a MOI of 5, and cell supernatants were collected at indicated time points for analysis of the concentration of IFN-β by ELISA. (B) A549 cells were co-transfected with pISRE-TA-Luc and RLTK, and the cells were then infected with MPC/04 and B/03 at a MOI of 5 at 12 h post-transfection. Relative luciferase activities were determined at different time points after reovirus infection using a luciferase assay kit. Sendai virus (SeV) infection was included as a positive control, and its relative luciferase activity was determined at 12 h post-infection. Transfection but no stimulation served as a mock control. (C) A549 cells were co-transfected with pISRE-TA-Luc and RLTK or shAkt or shCtrl, and the cells were treated with anti-IFNAR1 and -IFN lambda R1 antibodies or without antibodies 12 h after transfection. 2 h after treatment, cells were infected with MPC/04 and B/03 at a MOI of 5 or SeV. Relative luciferase activities were determined at 24 h post infection using a luciferase assay kit. (D) Vero cells were transfected with 4 μg/well of shAkt, transfected with 4 μg/well of shCtrl or left untransfected for 24 h, and the cells were then infected with MPC/04 and B/03. After a 1 h incubation, the monolayers were covered with 2 ml of 1% Bacto Agar and 2 × MEM containing 10 μg/ml TLCK-treated α-CHT. Plates were photographed 4 days later for plaque counting. (E) Analysis of phosphorylated EMSY in MPC/04- and B/03-infected cells with and without LY294002 (50 μM) treatment at 30 min post-infection. (F) A549 cells were transfected with empty plasmid or a plasmid expressing EMSY, EMSY-targeting shRNA or control (shCtl). 24 h after transfection, the cells were infected with MPC/04 and B/03 at a MOI of 5, and viral titers of cell supernatants were determined 24 h after infection. (G) 12 h after A549 cells were cotransfected with pISRE-TA-Luc and RLTK, the cells pretreated with or without LY294002 (50 μM) were infected with MPC/04 and B/03 at a MOI of 5 or mock infected at 12 h post-transfection. SeV infection was included as a positive control. Relative luciferase activities were determined at 12 h p.i. using a luciferase assay kit. (H) A549 cells were cotransfected with pISRE-TA-Luc, RLTK and shAkt or shCtrl (negative control). 12 h after transfection, the cells were infected with MPC/04 and B/03 at a MOI of 5 or mock infected at 12 h post-transfection. SeV infection was included as a positive control. Relative luciferase activities were determined at 12 h p.i. using a luciferase assay kit. (I–K) Analysis of mRNA levels of IFITM1, ISG15 and Viperin in MPC/04- and B/03- infected cells with or without pre-treatment with LY294002 (50 μM) (I) or with transfection with shAkt or shCtrl (negative control) (J) or with transfection with shEMSY or shCtrl (negative control) (K) by qPCR. (L–N) 24 h after transfection with plasmids expressing IFITM1, ISG15, Viperin (L) , shCtl, ISG15-targeting shRNA (sh-ISG15) or Viperin-targeting shRNA (sh-Viperin) (M,N) , cells were infected with MPC/04 and B/03 at a MOI of 5, and the relative RNA levels of viral S4 gene (M) and viral titers of cell supernatants (L,N) were determined 24 h after infection. Data represent the results of three independent experiments. * p
Figure Legend Snippet: Role of IFN-stimulated genes in PI3K/AKT-mediated modulation of virus replication. (A) A549 cells were infected with MPC/04 and B/03 at a MOI of 5, and cell supernatants were collected at indicated time points for analysis of the concentration of IFN-β by ELISA. (B) A549 cells were co-transfected with pISRE-TA-Luc and RLTK, and the cells were then infected with MPC/04 and B/03 at a MOI of 5 at 12 h post-transfection. Relative luciferase activities were determined at different time points after reovirus infection using a luciferase assay kit. Sendai virus (SeV) infection was included as a positive control, and its relative luciferase activity was determined at 12 h post-infection. Transfection but no stimulation served as a mock control. (C) A549 cells were co-transfected with pISRE-TA-Luc and RLTK or shAkt or shCtrl, and the cells were treated with anti-IFNAR1 and -IFN lambda R1 antibodies or without antibodies 12 h after transfection. 2 h after treatment, cells were infected with MPC/04 and B/03 at a MOI of 5 or SeV. Relative luciferase activities were determined at 24 h post infection using a luciferase assay kit. (D) Vero cells were transfected with 4 μg/well of shAkt, transfected with 4 μg/well of shCtrl or left untransfected for 24 h, and the cells were then infected with MPC/04 and B/03. After a 1 h incubation, the monolayers were covered with 2 ml of 1% Bacto Agar and 2 × MEM containing 10 μg/ml TLCK-treated α-CHT. Plates were photographed 4 days later for plaque counting. (E) Analysis of phosphorylated EMSY in MPC/04- and B/03-infected cells with and without LY294002 (50 μM) treatment at 30 min post-infection. (F) A549 cells were transfected with empty plasmid or a plasmid expressing EMSY, EMSY-targeting shRNA or control (shCtl). 24 h after transfection, the cells were infected with MPC/04 and B/03 at a MOI of 5, and viral titers of cell supernatants were determined 24 h after infection. (G) 12 h after A549 cells were cotransfected with pISRE-TA-Luc and RLTK, the cells pretreated with or without LY294002 (50 μM) were infected with MPC/04 and B/03 at a MOI of 5 or mock infected at 12 h post-transfection. SeV infection was included as a positive control. Relative luciferase activities were determined at 12 h p.i. using a luciferase assay kit. (H) A549 cells were cotransfected with pISRE-TA-Luc, RLTK and shAkt or shCtrl (negative control). 12 h after transfection, the cells were infected with MPC/04 and B/03 at a MOI of 5 or mock infected at 12 h post-transfection. SeV infection was included as a positive control. Relative luciferase activities were determined at 12 h p.i. using a luciferase assay kit. (I–K) Analysis of mRNA levels of IFITM1, ISG15 and Viperin in MPC/04- and B/03- infected cells with or without pre-treatment with LY294002 (50 μM) (I) or with transfection with shAkt or shCtrl (negative control) (J) or with transfection with shEMSY or shCtrl (negative control) (K) by qPCR. (L–N) 24 h after transfection with plasmids expressing IFITM1, ISG15, Viperin (L) , shCtl, ISG15-targeting shRNA (sh-ISG15) or Viperin-targeting shRNA (sh-Viperin) (M,N) , cells were infected with MPC/04 and B/03 at a MOI of 5, and the relative RNA levels of viral S4 gene (M) and viral titers of cell supernatants (L,N) were determined 24 h after infection. Data represent the results of three independent experiments. * p

Techniques Used: Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transfection, Luciferase, Positive Control, Activity Assay, Incubation, Plasmid Preparation, Expressing, shRNA, Negative Control, Real-time Polymerase Chain Reaction

3) Product Images from "Production of Siderophores Increases Resistance to Fusaric Acid in Pseudomonas protegens Pf-5"

Article Title: Production of Siderophores Increases Resistance to Fusaric Acid in Pseudomonas protegens Pf-5

Journal: PLoS ONE

doi: 10.1371/journal.pone.0117040

Effect of FA on cell motility and biofilm formation by P. protegens Pf-5. A . Cultures of P. protegens Pf-5 (wild type) were grown aerobically overnight to OD = 3 in KMB medium. An aliquot was spotted onto the centre of plates containing 20 fold diluted KMB medium supplemented or not with FA and solidified with various concentrations of Bacto-Agar, depending on which motility was to be evaluated. After 24 h of incubation at room temperature, the distance of the migration front from the point of inoculation was measured. B . Cultures of P. protegens Pf-5 were grown in 96-well polystyrene plates containing E 2 glucose minimal medium supplemented or not with FA. After 24 h of incubation, biofilm formation was assessed by the determining the proportion of attached and non-attached bacteria (A 595 /OD 600 ). In all cases, error bars indicate the standard deviation of the mean. The asterisk (*) denotes significant differences (P
Figure Legend Snippet: Effect of FA on cell motility and biofilm formation by P. protegens Pf-5. A . Cultures of P. protegens Pf-5 (wild type) were grown aerobically overnight to OD = 3 in KMB medium. An aliquot was spotted onto the centre of plates containing 20 fold diluted KMB medium supplemented or not with FA and solidified with various concentrations of Bacto-Agar, depending on which motility was to be evaluated. After 24 h of incubation at room temperature, the distance of the migration front from the point of inoculation was measured. B . Cultures of P. protegens Pf-5 were grown in 96-well polystyrene plates containing E 2 glucose minimal medium supplemented or not with FA. After 24 h of incubation, biofilm formation was assessed by the determining the proportion of attached and non-attached bacteria (A 595 /OD 600 ). In all cases, error bars indicate the standard deviation of the mean. The asterisk (*) denotes significant differences (P

Techniques Used: Incubation, Migration, Standard Deviation

4) Product Images from "The post-translational modification of the Clostridium difficile flagellin affects motility, cell surface properties and virulence"

Article Title: The post-translational modification of the Clostridium difficile flagellin affects motility, cell surface properties and virulence

Journal: Molecular Microbiology

doi: 10.1111/mmi.12755

Motility in M68 putative modification mutants. Motility assays of both the 630Δ erm and M68Δ erm parental strains compared to the M68Δ fliC in-frame deletion mutant and the mutations in the predicted modification genes M68_CD0242 and M68_CD0243 were carried out in CDMM containing 0.3% Difco-bacto agar. M68Δ erm was motile and the M68_ fliC mutant non-motile, as described previously. A mutant of the M68_0242 gene was non-motile, this was restored upon complementation. The M68_0243 mutant was motile, with a slightly reduced motility compared to the parent.
Figure Legend Snippet: Motility in M68 putative modification mutants. Motility assays of both the 630Δ erm and M68Δ erm parental strains compared to the M68Δ fliC in-frame deletion mutant and the mutations in the predicted modification genes M68_CD0242 and M68_CD0243 were carried out in CDMM containing 0.3% Difco-bacto agar. M68Δ erm was motile and the M68_ fliC mutant non-motile, as described previously. A mutant of the M68_0242 gene was non-motile, this was restored upon complementation. The M68_0243 mutant was motile, with a slightly reduced motility compared to the parent.

Techniques Used: Modification, Mutagenesis

Motility and flagella production in 630 putative modification mutants.A. Motility assays of the 630Δ erm parental strain compared to the fliC Clostron mutant and the mutations in the predicted modification genes were carried out in CDMM containing 0.3% Difco-bacto agar. The fliC mutant and the initial GT mutant were non-motile, as described previously, as were mutations in the ORFs CD0241, CD0242 and CD0244. These were all restored to motility by complementation.B. Flagellin from 630Δ erm and the modification mutants were probed with an anti-FliC antibody by Western blot (MW = molecular weight, gene names and numbers below indicate mutation, comp = in trans complementation of mutant). All strains with the exception of the fliC mutant were found to produce flagellin, although the mass of the protein varied.C. The parental strain 630Δ erm and the mutants of CD0241, CD0242, CD0243 and CD0244 were observed by TEM, the black scale bar in each image represents a length of 1 μm. All of the mutants appeared to have flagella associated with the surface of the cells.
Figure Legend Snippet: Motility and flagella production in 630 putative modification mutants.A. Motility assays of the 630Δ erm parental strain compared to the fliC Clostron mutant and the mutations in the predicted modification genes were carried out in CDMM containing 0.3% Difco-bacto agar. The fliC mutant and the initial GT mutant were non-motile, as described previously, as were mutations in the ORFs CD0241, CD0242 and CD0244. These were all restored to motility by complementation.B. Flagellin from 630Δ erm and the modification mutants were probed with an anti-FliC antibody by Western blot (MW = molecular weight, gene names and numbers below indicate mutation, comp = in trans complementation of mutant). All strains with the exception of the fliC mutant were found to produce flagellin, although the mass of the protein varied.C. The parental strain 630Δ erm and the mutants of CD0241, CD0242, CD0243 and CD0244 were observed by TEM, the black scale bar in each image represents a length of 1 μm. All of the mutants appeared to have flagella associated with the surface of the cells.

Techniques Used: Modification, Mutagenesis, Western Blot, Molecular Weight, Transmission Electron Microscopy

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    Difco bacto agar
    DHHC20 expression induces transformation of NIH/3t3 cells. (A) Suspensions of each clone in exponential growth phase were plated in <t>DMEM</t> containing 0.33% <t>Bacto-Agar</t> overlaid in 35 mm plates with 0.6% agar gel. The cells were incubated for 21 days and
    Bacto Agar, supplied by Difco, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bacto agar/product/Difco
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bacto agar - by Bioz Stars, 2022-01
    86/100 stars
      Buy from Supplier

    86
    Difco difco bacto agar
    Precocious swarming of waaG LPS core mutant. (A) A wild-type (wt) S. enterica serovar Typhimurium strain (SL3770) and its waaG mutant derivative (SL3769) were inoculated on <t>Difco</t> <t>Bacto</t> swarm medium and incubated at 37°C. Photographs were taken after 5 and 20 h of incubation. The arrow points to edge of the waaG swarm colony. (B) The waaG strain was inoculated on minimal swarm medium and incubated at 37°C. Photographs were taken after 24, 30, and 44 h. The arrow indicates edge of the slime ring. Swarming movement was confined to regions that appear as branches emanating from the center.
    Difco Bacto Agar, supplied by Difco, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/difco bacto agar/product/Difco
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    difco bacto agar - by Bioz Stars, 2022-01
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    DHHC20 expression induces transformation of NIH/3t3 cells. (A) Suspensions of each clone in exponential growth phase were plated in DMEM containing 0.33% Bacto-Agar overlaid in 35 mm plates with 0.6% agar gel. The cells were incubated for 21 days and

    Journal: Molecular membrane biology

    Article Title: DHHC20: a human palmitoyl acyltransferase that causes cellular transformation

    doi: 10.3109/09687681003616854

    Figure Lengend Snippet: DHHC20 expression induces transformation of NIH/3t3 cells. (A) Suspensions of each clone in exponential growth phase were plated in DMEM containing 0.33% Bacto-Agar overlaid in 35 mm plates with 0.6% agar gel. The cells were incubated for 21 days and

    Article Snippet: Ten-thousand stably transfected human DHHC20 -expressing cells (DC1, DC3), DHHS20 -expressing cells (DS1, DS4), and non- DHHC20/DHHS20 -expressing cells (E1, E2) in exponential growth phase were plated in DMEM containing 10% bovine serum and 0.6% Bacto-Agar (Difco) in 35 mm plates on day 1 and incubated in the atmosphere noted above.

    Techniques: Expressing, Transformation Assay, Incubation

    Modulation of reovirus replication by the PI3K/Akt pathway. Serum-starved A549 cells were pre-incubated with increasing concentrations of LY294002, wortmannin or DMSO for 1 h, and they were then infected with MPC/04 and B/03 at a MOI of 5 in the presence of each drug for 24 h. The total RNA of the cells was prepared, and S4 gene expression was analyzed by qPCR (A) . Cells and cell supernatants were harvested together, and subjected to a single freeze/thaw step for analysis of viral titers (B) . Expression of Akt1 was determined by qPCR analysis and Western blot analysis using Akt1- and GAPDH-specific antibodies (C) . Effect of inhibition of Akt1 expression on virus replication was also evaluated by plaque sizes in A549 cells (D) . A549 cells were transfected with 4 μg/well of shAkt or shCtrl for 24 h, and the cells were then infected with MPC/04 and B/03. After 1 h incubation, the monolayers were covered with 2 ml of 1% Bacto Agar and 2 × MEM containing 10 μg/ml TLCK-treated α-CHT. Plaques were photographed 4 days later. (E) A549 cells were transfected with 2 μg/well of shAkt or shCtrl for 12 h, and the cells were then infected with MPC/04 or B/03 at a MOI of 5. Cell supernatants were harvested at the indicated time point and titrated on L929 cells for the plaque assay. The data shown in (A–C) and (E) represent the results of three independent experiments. * p

    Journal: Frontiers in Microbiology

    Article Title: Blocking the PI3K/AKT pathway enhances mammalian reovirus replication by repressing IFN-stimulated genes

    doi: 10.3389/fmicb.2015.00886

    Figure Lengend Snippet: Modulation of reovirus replication by the PI3K/Akt pathway. Serum-starved A549 cells were pre-incubated with increasing concentrations of LY294002, wortmannin or DMSO for 1 h, and they were then infected with MPC/04 and B/03 at a MOI of 5 in the presence of each drug for 24 h. The total RNA of the cells was prepared, and S4 gene expression was analyzed by qPCR (A) . Cells and cell supernatants were harvested together, and subjected to a single freeze/thaw step for analysis of viral titers (B) . Expression of Akt1 was determined by qPCR analysis and Western blot analysis using Akt1- and GAPDH-specific antibodies (C) . Effect of inhibition of Akt1 expression on virus replication was also evaluated by plaque sizes in A549 cells (D) . A549 cells were transfected with 4 μg/well of shAkt or shCtrl for 24 h, and the cells were then infected with MPC/04 and B/03. After 1 h incubation, the monolayers were covered with 2 ml of 1% Bacto Agar and 2 × MEM containing 10 μg/ml TLCK-treated α-CHT. Plaques were photographed 4 days later. (E) A549 cells were transfected with 2 μg/well of shAkt or shCtrl for 12 h, and the cells were then infected with MPC/04 or B/03 at a MOI of 5. Cell supernatants were harvested at the indicated time point and titrated on L929 cells for the plaque assay. The data shown in (A–C) and (E) represent the results of three independent experiments. * p

    Article Snippet: Briefly, samples were diluted and inoculated into 6-well plates of cells by incubation for 1 h. The monolayers were washed with 2 mL of phosphate-buffered saline (PBS) and covered with 2 mL of serum-free medium 199 (Irvine Scientific) and 1% Bacto Agar (DIFCO) containing 10 μg/mL TLCK-treated α-CHT (Sigma-Aldrich).

    Techniques: Incubation, Infection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Inhibition, Transfection, Plaque Assay

    Role of IFN-stimulated genes in PI3K/AKT-mediated modulation of virus replication. (A) A549 cells were infected with MPC/04 and B/03 at a MOI of 5, and cell supernatants were collected at indicated time points for analysis of the concentration of IFN-β by ELISA. (B) A549 cells were co-transfected with pISRE-TA-Luc and RLTK, and the cells were then infected with MPC/04 and B/03 at a MOI of 5 at 12 h post-transfection. Relative luciferase activities were determined at different time points after reovirus infection using a luciferase assay kit. Sendai virus (SeV) infection was included as a positive control, and its relative luciferase activity was determined at 12 h post-infection. Transfection but no stimulation served as a mock control. (C) A549 cells were co-transfected with pISRE-TA-Luc and RLTK or shAkt or shCtrl, and the cells were treated with anti-IFNAR1 and -IFN lambda R1 antibodies or without antibodies 12 h after transfection. 2 h after treatment, cells were infected with MPC/04 and B/03 at a MOI of 5 or SeV. Relative luciferase activities were determined at 24 h post infection using a luciferase assay kit. (D) Vero cells were transfected with 4 μg/well of shAkt, transfected with 4 μg/well of shCtrl or left untransfected for 24 h, and the cells were then infected with MPC/04 and B/03. After a 1 h incubation, the monolayers were covered with 2 ml of 1% Bacto Agar and 2 × MEM containing 10 μg/ml TLCK-treated α-CHT. Plates were photographed 4 days later for plaque counting. (E) Analysis of phosphorylated EMSY in MPC/04- and B/03-infected cells with and without LY294002 (50 μM) treatment at 30 min post-infection. (F) A549 cells were transfected with empty plasmid or a plasmid expressing EMSY, EMSY-targeting shRNA or control (shCtl). 24 h after transfection, the cells were infected with MPC/04 and B/03 at a MOI of 5, and viral titers of cell supernatants were determined 24 h after infection. (G) 12 h after A549 cells were cotransfected with pISRE-TA-Luc and RLTK, the cells pretreated with or without LY294002 (50 μM) were infected with MPC/04 and B/03 at a MOI of 5 or mock infected at 12 h post-transfection. SeV infection was included as a positive control. Relative luciferase activities were determined at 12 h p.i. using a luciferase assay kit. (H) A549 cells were cotransfected with pISRE-TA-Luc, RLTK and shAkt or shCtrl (negative control). 12 h after transfection, the cells were infected with MPC/04 and B/03 at a MOI of 5 or mock infected at 12 h post-transfection. SeV infection was included as a positive control. Relative luciferase activities were determined at 12 h p.i. using a luciferase assay kit. (I–K) Analysis of mRNA levels of IFITM1, ISG15 and Viperin in MPC/04- and B/03- infected cells with or without pre-treatment with LY294002 (50 μM) (I) or with transfection with shAkt or shCtrl (negative control) (J) or with transfection with shEMSY or shCtrl (negative control) (K) by qPCR. (L–N) 24 h after transfection with plasmids expressing IFITM1, ISG15, Viperin (L) , shCtl, ISG15-targeting shRNA (sh-ISG15) or Viperin-targeting shRNA (sh-Viperin) (M,N) , cells were infected with MPC/04 and B/03 at a MOI of 5, and the relative RNA levels of viral S4 gene (M) and viral titers of cell supernatants (L,N) were determined 24 h after infection. Data represent the results of three independent experiments. * p

    Journal: Frontiers in Microbiology

    Article Title: Blocking the PI3K/AKT pathway enhances mammalian reovirus replication by repressing IFN-stimulated genes

    doi: 10.3389/fmicb.2015.00886

    Figure Lengend Snippet: Role of IFN-stimulated genes in PI3K/AKT-mediated modulation of virus replication. (A) A549 cells were infected with MPC/04 and B/03 at a MOI of 5, and cell supernatants were collected at indicated time points for analysis of the concentration of IFN-β by ELISA. (B) A549 cells were co-transfected with pISRE-TA-Luc and RLTK, and the cells were then infected with MPC/04 and B/03 at a MOI of 5 at 12 h post-transfection. Relative luciferase activities were determined at different time points after reovirus infection using a luciferase assay kit. Sendai virus (SeV) infection was included as a positive control, and its relative luciferase activity was determined at 12 h post-infection. Transfection but no stimulation served as a mock control. (C) A549 cells were co-transfected with pISRE-TA-Luc and RLTK or shAkt or shCtrl, and the cells were treated with anti-IFNAR1 and -IFN lambda R1 antibodies or without antibodies 12 h after transfection. 2 h after treatment, cells were infected with MPC/04 and B/03 at a MOI of 5 or SeV. Relative luciferase activities were determined at 24 h post infection using a luciferase assay kit. (D) Vero cells were transfected with 4 μg/well of shAkt, transfected with 4 μg/well of shCtrl or left untransfected for 24 h, and the cells were then infected with MPC/04 and B/03. After a 1 h incubation, the monolayers were covered with 2 ml of 1% Bacto Agar and 2 × MEM containing 10 μg/ml TLCK-treated α-CHT. Plates were photographed 4 days later for plaque counting. (E) Analysis of phosphorylated EMSY in MPC/04- and B/03-infected cells with and without LY294002 (50 μM) treatment at 30 min post-infection. (F) A549 cells were transfected with empty plasmid or a plasmid expressing EMSY, EMSY-targeting shRNA or control (shCtl). 24 h after transfection, the cells were infected with MPC/04 and B/03 at a MOI of 5, and viral titers of cell supernatants were determined 24 h after infection. (G) 12 h after A549 cells were cotransfected with pISRE-TA-Luc and RLTK, the cells pretreated with or without LY294002 (50 μM) were infected with MPC/04 and B/03 at a MOI of 5 or mock infected at 12 h post-transfection. SeV infection was included as a positive control. Relative luciferase activities were determined at 12 h p.i. using a luciferase assay kit. (H) A549 cells were cotransfected with pISRE-TA-Luc, RLTK and shAkt or shCtrl (negative control). 12 h after transfection, the cells were infected with MPC/04 and B/03 at a MOI of 5 or mock infected at 12 h post-transfection. SeV infection was included as a positive control. Relative luciferase activities were determined at 12 h p.i. using a luciferase assay kit. (I–K) Analysis of mRNA levels of IFITM1, ISG15 and Viperin in MPC/04- and B/03- infected cells with or without pre-treatment with LY294002 (50 μM) (I) or with transfection with shAkt or shCtrl (negative control) (J) or with transfection with shEMSY or shCtrl (negative control) (K) by qPCR. (L–N) 24 h after transfection with plasmids expressing IFITM1, ISG15, Viperin (L) , shCtl, ISG15-targeting shRNA (sh-ISG15) or Viperin-targeting shRNA (sh-Viperin) (M,N) , cells were infected with MPC/04 and B/03 at a MOI of 5, and the relative RNA levels of viral S4 gene (M) and viral titers of cell supernatants (L,N) were determined 24 h after infection. Data represent the results of three independent experiments. * p

    Article Snippet: Briefly, samples were diluted and inoculated into 6-well plates of cells by incubation for 1 h. The monolayers were washed with 2 mL of phosphate-buffered saline (PBS) and covered with 2 mL of serum-free medium 199 (Irvine Scientific) and 1% Bacto Agar (DIFCO) containing 10 μg/mL TLCK-treated α-CHT (Sigma-Aldrich).

    Techniques: Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transfection, Luciferase, Positive Control, Activity Assay, Incubation, Plasmid Preparation, Expressing, shRNA, Negative Control, Real-time Polymerase Chain Reaction

    Effect of FA on cell motility and biofilm formation by P. protegens Pf-5. A . Cultures of P. protegens Pf-5 (wild type) were grown aerobically overnight to OD = 3 in KMB medium. An aliquot was spotted onto the centre of plates containing 20 fold diluted KMB medium supplemented or not with FA and solidified with various concentrations of Bacto-Agar, depending on which motility was to be evaluated. After 24 h of incubation at room temperature, the distance of the migration front from the point of inoculation was measured. B . Cultures of P. protegens Pf-5 were grown in 96-well polystyrene plates containing E 2 glucose minimal medium supplemented or not with FA. After 24 h of incubation, biofilm formation was assessed by the determining the proportion of attached and non-attached bacteria (A 595 /OD 600 ). In all cases, error bars indicate the standard deviation of the mean. The asterisk (*) denotes significant differences (P

    Journal: PLoS ONE

    Article Title: Production of Siderophores Increases Resistance to Fusaric Acid in Pseudomonas protegens Pf-5

    doi: 10.1371/journal.pone.0117040

    Figure Lengend Snippet: Effect of FA on cell motility and biofilm formation by P. protegens Pf-5. A . Cultures of P. protegens Pf-5 (wild type) were grown aerobically overnight to OD = 3 in KMB medium. An aliquot was spotted onto the centre of plates containing 20 fold diluted KMB medium supplemented or not with FA and solidified with various concentrations of Bacto-Agar, depending on which motility was to be evaluated. After 24 h of incubation at room temperature, the distance of the migration front from the point of inoculation was measured. B . Cultures of P. protegens Pf-5 were grown in 96-well polystyrene plates containing E 2 glucose minimal medium supplemented or not with FA. After 24 h of incubation, biofilm formation was assessed by the determining the proportion of attached and non-attached bacteria (A 595 /OD 600 ). In all cases, error bars indicate the standard deviation of the mean. The asterisk (*) denotes significant differences (P

    Article Snippet: Briefly, 0.005 mL of the culture was spotted at the center of five replicate plates containing 20-fold diluted KMB with or without FA, and 0.3% and 0.6% (wt/vol) Bacto-Agar (Difco) for the evaluation of swimming motility and determination of swarming motility, respectively.

    Techniques: Incubation, Migration, Standard Deviation

    Precocious swarming of waaG LPS core mutant. (A) A wild-type (wt) S. enterica serovar Typhimurium strain (SL3770) and its waaG mutant derivative (SL3769) were inoculated on Difco Bacto swarm medium and incubated at 37°C. Photographs were taken after 5 and 20 h of incubation. The arrow points to edge of the waaG swarm colony. (B) The waaG strain was inoculated on minimal swarm medium and incubated at 37°C. Photographs were taken after 24, 30, and 44 h. The arrow indicates edge of the slime ring. Swarming movement was confined to regions that appear as branches emanating from the center.

    Journal: Journal of Bacteriology

    Article Title: Genetics of Swarming Motility in Salmonella enterica Serovar Typhimurium: Critical Role for Lipopolysaccharide

    doi:

    Figure Lengend Snippet: Precocious swarming of waaG LPS core mutant. (A) A wild-type (wt) S. enterica serovar Typhimurium strain (SL3770) and its waaG mutant derivative (SL3769) were inoculated on Difco Bacto swarm medium and incubated at 37°C. Photographs were taken after 5 and 20 h of incubation. The arrow points to edge of the waaG swarm colony. (B) The waaG strain was inoculated on minimal swarm medium and incubated at 37°C. Photographs were taken after 24, 30, and 44 h. The arrow indicates edge of the slime ring. Swarming movement was confined to regions that appear as branches emanating from the center.

    Article Snippet: This view is also supported by our inability to demonstrate a dialyzable constituent in the Eiken agar that promotes swarming on Difco Bacto agar (our unpublished results).

    Techniques: Mutagenesis, Incubation